The Biodiscovery Platform – Exploring synthetic microbiomes

Case 1: Synthetic human gut microbiomes

Background:

There is increasing interest in developing synthetic microbial communities for answering fundamental questions as well as developing a broad range of biotechnological applications. With access to multi-omics technologies as well as innovations in microbial cultivation, there is a potential for predicting and experimentally validating metabolic interaction networks and their influence on stability and functioning of synthetic communities. The 16S rRNA amplicon data analysis procedures can be fine-tuned based on information of candidate strains such as the number of 16S rRNA gene copies/genome and using custom databases with known copies of 16S rRNA gene sequences. Moreover, application of (meta)transcriptomics and (meta)proteomics can aid in identifying strain specific active contribution guiding in targeted engineering of bioprocesses. However, until now integration of multi-omics approaches to understand and engineer microbial communities and functions has been a major challenge. With in-house expertise in microbial ecology, physiology and genomics, our team at UNLOCK can assist in both design and comprehensive characterization of synthetic microbial communities for researchers. Furthermore, we can predict, validate and optimize the contribution of individual strains to functional traits within the community. 

Case study

At MIB-WUR, we have extensive experience in microbial ecology, physiology and functional (meta)genomics. We have isolated a broad range of novel anaerobic bacteria from diverse ecosystems, one of them being the human gut. A synthetic gut microbiome has recently been designed for reconstructing the central metabolic pathway, unravelling inter-species metabolic interactions and to identify contribution of key gut bacteria to a multitude of functions related to degradation of complex carbon sources and production of short chain fatty acids (SCFA). The knowledge of specific metabolic roles of core gut bacteria can aid in targeted engineering of the human gut microbiome for health benefits.

Approach

We first screened >5000 shotgun metagenomics datasets from public databases to identify highly prevalent core bacterial species in the human gut. We shortlisted 10 core gut bacteria using a combination of genome-based prediction, in-silico metabolic network-based predictions of competition and complementarity, and the available information of physiology of core bacteria. The design was aimed towards efforts to a) Reconstruct the central metabolic pathway from diet to SCFA production; b) Identify metabolic basis for co-existence of competing core species; c) Identify contribution of each strain to multitude of functions related to degradation and SCFA production.

Figure 1: Overview of process from data mining to design and investigation of synthetic gut microbiomes for unravelling community stability and functional processes.

The workflow from design to data analysis is shown in figure 1. We carried out metabolite measurements, quantitative microbiota profiling and metatranscriptomics. Three-way integration and identification of key ecological and functional aspects of the synthetic gut microbiome can be achieved through our UNLOCK FAIR data platform. The UNLOCK FAIR data platform consist of bioinformatics pipelines which process raw data and generate output for species abundances as well as active gene/pathway abundances that can be further analysed together with metabolite data using our custom workflows provided as Jupyter Notebooks.

Case 2: A synthetic microbial community for syngas fermentation

Background

Microorganisms have been used in biotechnological processes already for centuries. Two scenarios are often applied: mixed culture (open) cultivation (e.g. in food fermentations, wastewater treatment), or pure culture cultivation. In pure culture cultivations, genetic engineering of the microbes is often applied to, for example, overproduce a metabolite or introduce new pathways for the production of non-native compounds. An option that is much less explored is the design of defined synthetic co-cultures – such an approach can pave new ways in biotechnology as one can think of several microbial networks for the production of compounds that are not easily produced by a single microbe or open cultures of microbes.

Case study

We previously designed a synthetic culture that can convert syngas to medium-chain fatty acids (MCFA) and/or elongated acids (Diender et al., 2016). This co-culture is composed of an acetogenic bacterium, Clostridium autoethanogenum, and a chain-elongator, Clostridium kluyveri. Carbon monoxide (and H2 plus CO2) present in syngas can be converted to acetate and ethanol by C. autoethanogenum, and these two compounds serve as the substrate for C. kluyveri to produce MCFA. With the expertise of the team at UNLOCK we can further study the interaction of these two microorganisms, but also design and test more complex networks for improved MCFA production or increased range of products (for example, production of odd-chain MCFA). 

Approach

We operated parallel chemostat reactors with a pure culture of C. autoethanogenum and a synthetic co-cultures of C. autoethanogenum plus C. kluyveri. These chemostats were fed with CO, and CO + H2. The objective was to get insights into the interactions between the two microorganisms, and apply this knowledge to further optimize the performance of the co-culture. Cultures’ performance was studied by measuring metabolites during fermentation. An interesting outcome of the work was the increased flow of electrons towards ethanol by C. autoethanogenum, when co-cultivated with C. kluyveri (Figure 1). This was linked to the sink for ethanol created by the presence of C. kluyveri, and this is beneficial for both microorganisms in the co-culture.

Figure 1: Electron flow in monocultures of C. autoethanogenum and co-cultures of C. autoethanogenum and C. kluyveri fed with CO or CO+H2 in a chemostat.

We further analysed the transcriptome of C. autoethanogenum in pure and synthetic co-cultures. Expression of the central carbon- and energy-metabolism remained unaltered during co-cultivation. In contrast, increased expression of genes related to amino acid synthesis, pili/flagella formation/assembly and antibiotics production was observed during co-cultivation. Furthermore, striking changes in metal import related genes for iron and molybdenum were also observed.

We further analysed the transcriptome of C. autoethanogenum in pure and synthetic co-cultures. Expression of the central carbon- and energy-metabolism remained unaltered during co-cultivation. In contrast, increased expression of genes related to amino acid synthesis, pili/flagella formation/assembly and antibiotics production was observed during co-cultivation. Furthermore, striking changes in metal import related genes for iron and molybdenum were also observed.